Zhu Xuexue, Ma Ke, Zhou Kuo, Pan Xia, Liu Jibin, Nürnberg Bernd, Alesutan Ioana, Völkl Jakob, Lang Florian
Department of Pharmacology, Experimental Therapy & Toxicology, Eberhard-Karls-University of Tübingen, Tübingen, Germany.
School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Kidney Blood Press Res. 2022;47(6):399-409. doi: 10.1159/000524050. Epub 2022 Mar 25.
BACKGROUND/AIMS: Vasopressin is a powerful stimulator of vascular calcification, augmenting osteogenic signaling in vascular smooth muscle cells (VSMCs) including upregulation of transcription factors such as core-binding factor α-1 (CBFA1), msh homeobox 2 (MSX2), and SRY-Box 9 (SOX9), as well as of tissue-nonspecific alkaline phosphatase (ALPL). Vasopressin-induced osteogenic signaling and calcification require the serum- and glucocorticoid-inducible kinase 1 (SGK1). Known effects of SGK1 include upregulation of Na+/H+ exchanger 1 (NHE1). NHE1 further participates in the regulation of reactive oxygen species (ROS). NHE1 has been shown to participate in the orchestration of bone mineralization. The present study, thus, explored whether vasopressin modifies NHE1 expression and ROS generation, as well as whether pharmacological inhibition of NHE1 disrupts vasopressin-induced osteogenic signaling and calcification in VSMCs.
Human aortic smooth muscle cells (HAoSMCs) were treated with vasopressin in the absence or presence of SGK1 silencing, SGK1 inhibitor GSK-650394, and NHE1 blocker cariporide. Transcript levels were determined by using quantitative real-time polymerase chain reaction, protein abundance by Western blotting, ROS generation with 2',7'-dichlorofluorescein diacetate fluorescence, and ALP activity and calcium content by using colorimetric assays.
Vasopressin significantly enhanced the NHE1 transcript and protein levels in HAoSMCs, effects significantly blunted by SGK1 inhibition with GSK-650394 or SGK1 silencing. Vasopressin increased ROS accumulation, an effect significantly blocked by the NHE1 inhibitor cariporide. Vasopressin further significantly increased osteogenic markers CBFA1, MSX2, SOX9, and ALPL transcript levels, as well as ALP activity and calcium content in HAoSMCs, all effects significantly blunted by SGK1 silencing or in the presence of GSK-650394 or cariporide.
Vasopressin stimulates NHE1 expression and ROS generation, an effect dependent on SGK1 and required for vasopressin-induced stimulation of osteogenic signaling and calcification of VSMCs.
背景/目的：血管加压素是血管钙化的有力刺激因子，可增强血管平滑肌细胞（VSMC）中的成骨信号，包括上调转录因子如核心结合因子α-1（CBFA1）、msh同源盒2（MSX2）和SRY盒9（SOX9），以及组织非特异性碱性磷酸酶（ALPL）。血管加压素诱导的成骨信号和钙化需要血清和糖皮质激素诱导激酶1（SGK1）。SGK1的已知作用包括上调钠/氢交换体1（NHE1）。NHE1进一步参与活性氧（ROS）的调节。NHE1已被证明参与骨矿化的调控。因此，本研究探讨了血管加压素是否会改变NHE1表达和ROS生成，以及NHE1的药理学抑制是否会破坏血管加压素诱导的VSMC成骨信号和钙化。
在不存在或存在SGK1沉默、SGK1抑制剂GSK-650394和NHE1阻滞剂卡里波罗的情况下，用人血管加压素处理人主动脉平滑肌细胞（HAoSMC）。通过定量实时聚合酶链反应测定转录水平，通过蛋白质印迹法测定蛋白质丰度，用2',7'-二氯荧光素二乙酸酯荧光法测定ROS生成，用比色法测定ALP活性和钙含量。
血管加压素显著提高了HAoSMC中NHE1的转录水平和蛋白质水平，GSK-650394抑制SGK1或沉默SGK1可显著减弱这些作用。血管加压素增加了ROS积累，NHE1抑制剂卡里波罗可显著阻断这一作用。血管加压素进一步显著提高了HAoSMC中成骨标志物CBFA1、MSX2、SOX9和ALPL的转录水平，以及ALP活性和钙含量，SGK1沉默或存在GSK-650394或卡里波罗时，所有这些作用均显著减弱。
血管加压素刺激NHE1表达和ROS生成，这一作用依赖于SGK1，并且是血管加压素诱导的VSMC成骨信号刺激和钙化所必需的。
